Analytical chemistry

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Analytical chemistry (分析化学)

Mount Usu / Sarobetsu post-mined peatland
From left: Crater basin in 1986 and 2006. Cottongrass / Daylily

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[ protocol | unit | culture | validation ]

Analytical purposes

Quantitative analysis ≈ measurement

Lord Kelvin (1824-1907) "If you cannot measure, your knowledge is meager and unsatisfactory."

Qualitative analysis
State analysis

Objects

Inorganic analysis
Organic analysis

Absolute amount of samples

Macro analysis: 0.1-several g
Semi-micro analysis: 10-100 mg
Micro analysis: 1-10 mg
Ultra-micro analysis: < 1 mg

Relative amount of objects

Macro determination: 100ppm-100%
Major constituent determination: 1-100%
Minor constituent determination: 0.01-1%
Micro/trace determination: < 100ppm

Chemical microsensors and microinstrumentation

CHEMFETS (ISFET): Merits = small size (e.g., less than 1 mm² area), low output impedance, in vivo monitoring, small sample volumes, multiple-ion sensor arrays
The CHEMIST is essentially a conventional insulated-gate field effect transistor that has its metallic gate contact replaced by a chemically sensitive coating and a reference electrode.

Ion-controlled diodes

This device is a combination of a p-n junction and a metal oxide semiconductor capacitor in which the junction makes contact with the inversion layer of the capacitor. Like CHEMFET, the device can be use as an ion sensor by application of a suitable ion-selective coating to the gate surface.

Schottky diodes

In its simplest form Schottky diode consists of a small area of metal contact with a semiconductor. This contact exhibits a rectifying behavior and a characteristic nonlinear current-voltage dependence.

Chemresistors

In the case of organic semiconductors the use of such compounds as vapor sensors has been hindered by the rather high resistivities of the material (e.g., 10⁸-10¹⁵ ohm·cm).

Thin-film tin oxide gas sensors

Tin oxide and other metal oxide semiconductor are sensitive to low concentration of vapors. In operation a sintered block of the semiconductor is heated to several hundred degrees centigrade and its electrical conductivity is monitored.

Microdielectrometer

The device, presently manufactured by Micromet, consists of a planer interdigital microelectrode array, one side of which is attached as a floating gate to an onchip FET charge amplifier. The other side of the microelectrode array is driven with a sinusoidal voltage. By measuring the amplitude and phase differences of the signal applied to the driven gate and the signal produced by floating gate, it is possible to determine the complex impedance (i.e., the resistance and capacitance) of the medium in contact with the electrode. The integration of the FET amplifier and microelectrode permits extremely weak currents to be measured easily.

Porous carbon packings for liquid chromatography

Table 1. Comparison of properties of carbon and reversed-phase silica packings

Property	Reversed-phased silica	Carbon
Surface functional groups	n-Alkyl, alkylaryl, aryl, residual hydroxyis	Aromatic-type acidic and basic functional groups
Specific surface area	100-200 m²/g	5-1000 m²/g
Type and extent of particle porosity	Open pores 60-80%	Open and closed pores 30-60%
Mean pore size and pore size distribution	5-25 nm homogeneous mesopores	1-1000 nm heterogeneous micro-, meso-, macropores

significant figures or effective digits (有効数字, sf)

Digits being effective to its measurement resolution

Protocol (プロトコル)

A defined procedures, that can be assessed by anybody, for experiments and measurements.

phospholipid fatty acids
phosphorus

UV-absorbing compound concentration
staining of mycorrhizal fungi (菌根菌染色)

Chromatography (クロマトグラフィ)

Clinical analysis (臨床分析)

The improvement of sensitivity and specificity on chromatography

[ultraspure substances]__[simple introduction]
____|________________↓
____|________________[ionization/mass separation]
____|________________[dispersion convergence/detection]
____|________________↓
[standard spectrum]____ [mass spectrum analysis]

Fig. Connection to mass spectrometer

Target
analyte_Analyte
__A*____AA
__A*____AA
______+_________⇔____A*A______AA__+__A*A
_____YY_______________Y________Y
___Antibody_________Bound analyte________Free analyte

Fig. 3. Immunoassay test principle. The immunoassay reaction is a result of competition between tagged analyte (A^*) and unlabeled analyte (A) for a limited number of binding sites on the antibody (Y). As a result of the competitive reaction, the tagged analyte is partitioned between the fraction bound to antibody and the fraction free in solution.

(Chait & Ebersole 1981)

[ Markers on litter decomposition ]

Phospholipid fatty acids (PLFAs)

Protocol for the quantification

(Bligh & Dyer 1959)

Extraction of total lipids

5 g soil (powderization, if required)
↓ + 21:0 (internal standard material, I.S.) in benzene solution
↓ + 10 ml methanol
↓ + 5 ml chloroform
↓ + 4 ml water or buffer solution (citric acid or phosphoric acid)
∇ vortex (5 min)
∇ leave at rest (5 min)
↓ + 5 ml chloroform
∇ vortex (1 min)
↓ + 5 ml water or buffer solution (1:1:0.9)
∇ vortex
∇ 3000 rpm for 5 min.

In the test tube
H₂O/Methanol fraction
Chloroform fraction ← recover lipid by a Pasteur pipette
Debris (soil/litter)

↓ + 5 ml chloroform
∇ vortex
∇ ← recover chloroform fraction
↓ put the fractions together
total lipid

Separation of PLFAs from total lipids

chloroform fraction (= total lipid)
silicic acid chromatography
↓methanolysis + 2nd I.S.
↓GC
total fatty acid/g soil

Identification and quantification of PFLAs

If molecular species can not be identified, the species is determined by a gas chromatograph mass spectrometer (so-called GC mass)

Analysis of lipids by thin layer chromatography (TLC)

Preparation

Plate: silica gel wed. 0.25 mm (5 × 20 cm) Merk
Materials: purified lipid standard substance: GL (Cardiolypin), PG (Phosphatidyl glycerol), PE (Phosphatidyl ethanolamine)
Developing liquid

1) chloroform:methanol:water = 65:25:5
2) chloroform:methanol:acetone:acetic acid:water = 65:10:26:10:3

Reagent for detection: iodine, Dittner reagent, ninhydrin reagent

Manipulation

Spreading
make a small pot (φ = 20-80 mm) of 60-100 ml sample at 1.5 cm from the bottom of plate by a capillary tube
place 60-100 m solvent for 1-2 hr in a developing cabinet (to equilibrate the inisde)
Start development
prop the plate in a developing cabinet and leave them for 1.5-2 hours →

when the developing sample reaches to the 2-3 cm from the top, the developing is terminated

remove the plate from the cabinet and the solvent on the plate is diffused. Then, expose the plate to iodine →

whenk spot is found out, mark it! (principle = iodine addition)

Detection (I)

fill excess iodine crystal up a desiccator of which inside is saturated with steamed iodine
prop the plate against the wall of desiccator for a few minutes
lipids become yellow/brown by absorbing iodine → mark the spots by a pencil immediately after staining (to avoid color deterioration)

Detection (II) Ditter reagent = using for detecting phospholipids (The operations a and b must be undertaken under a draft chamber)

boil and gently add 4.01-g Mo₃ to 100-ml 25N H₂SO₄ (A)
add 0.18-g powdered Mo gently to 20-ml (A) for 15 minutes
collect the supernatant (B) by decantation after open cooling
mix equivalent amount of (A) and (B), and then add water of which amount is double of the mixture reagent
squirt the reagent to the plate → phospholipids are stained in blue

Detection (III). Ninhydrin reagent, used to detect amino acids

solve 0.25 g ninhydrin into acetone-lutidine solution (9:1)
spray the solution → leave it at 120°C for a few minutes → amino lipids are stained in burgundy (reddish violet)

Before the measurements (if possible), Rf on each spot should be calculated. Phospholipid used as the standard is run at the same time.
Cf. Extract component: phospholipids (90%), glycocide, fatty acids, etc.

Examples on the results

1st. PG was clearly distinguished. However, PG and CL were not clearly identified, due to the two spots of CL standard. (BR = brown-red)

    Sample     Line   Spot  Length Rf     Dittmer  Nin.

    Stand.     17.1   PE    6.95   0.406  blue     BR
                      PG    4.45   0.260  blue     blue
                      CL-x1 4.75   0.278  blue     blue
                      CL-x2 3.25   0.190  yellow   yellow
    20C left   16.95  a1    7.00   0.413  blue     BR
                      a2    4.25   0.251  blue     blue
                      a3    3.475  0.205  blue     blue
    20C right  16.95  b1    6.60   0.389  blue     BR
                      b2    3.95   0.233  blue     blue
                      b3    3.25   0.192  blue     blue
    37C        16.95  c1    6.70   0.395  blue     BR
                      c2    4.375  0.258  blue     blue
                      c3    3.45   0.204  blue     blue

2nd The separations are insufficient. In particular, there were a few unclear spots made by ninhydrin reaction

    Sample   Stand.            20C left    20C right   37C

    Line     16.4              16.55
    Spot     PE    PG    CL    d1    d2    e1    e2    f1    f2
    Length   4.075 3.35  4.05  4.05  3.2   3.9   3.05  3.95  3.05
    Rf       0.248 0.202 0.247 0.245 0.193 0.236 0.184 0.239 0.184
    Dittmer  blue  blue  blue  blue  blue  blue  blue  blue  blue
    Nin.     -     -     -     -     -     -     -     -     -

Extraction and fractionation (抽出・分画)

Centrifugation (遠心分離)

= centrifugal separation
Principle: separation by using density difference - not by the mass
i) centrifugal force, f_r

f_r = mrω²

m: particle mass (kg)
r = radial distance from center point (m)
ω = angular velocity = dθ/dt (rad/s)

G: relative centrifugal force, RCF = mrω²/mg = rω²/g
N: revolutions per minute, rpm

Centrifugal (遠心分離器)

= centrifugal machine, centrifugal precipitator, centrifugal separator, centrifuge, centrifuge machine or centrifuge separator
Swing-out roter 10⁴/min_______Fixed angled roter: 2 × 10⁵/min

centrifuge ___
r_max - r_min_______________r_max - r_min

Suspension: the mixed material added into the centrifuge tube
Pellet (precipitate): hard-packed concentration of particles after centrifugation
Supernatant: clarified liquid above the precipitate

Separation of ER

homogenatet cells
┃ centrigugate with 800-1000 × g, 5-10' + 0.2-0.4 M sucrose
┣━━━━━━━━━━━━━━━━┓_____⇑ not break organella
ppt (nuclei, chloroplast)_____supernatant
┏━━━━━━━━━━━━━━━━┫ 10000-15000 × g, 15-20'
ppt (mitochondria)_________supernatant = post-mitochondrial fraction
┏━━━━━━━━━━━━━━━━┫ 24000-40000 × g, 20-30'
ppt (r-ER + microsome)_____supernatant
┏━━━━━━━━━━━━━━━━┫ ultracentrifuge with
┃━━━━━━━━━━━━━━━━┃ 102000-105000 × g, 2 hr
ppt (free ribosomes polysomes) supernatant = S100 fraction
Furthermore, ribosomes are separated from r-ER by the separation the addtion of deoxycholate-N (DOC), Nonidet P40 (NP40), polyethylene cethylethen (Bry 58) or Triton-X100 that uncouples lipids in r-ER
*: g vs rpm = revolution per minutes
S: unit of centrifugal separation

Proteins (タンパク質)

Def. Isoelectric point (等電点), pI: 0 charge = net positive charge

Application:
Isoelectric focusing (等電点電気泳動), IEF
Isoelectric precipitation (等電点沈殿)

Purification and assay of enzymes (酵素の精製と分析)

D-Glycerate dehydrogenese purified from spinach leaves (EC 1.1.1.29 D-Glycerate NAD oxidoreductase)
COOH-CHOH-CH2OH (D-glycerate) + NAD⁺ ⇔ COOH-CO-CH2OH (Hydroxypyruvate) + NADH + H⁺

Aims

Figure out enzyme purification techniques and enzyme characteristics.

I. Purification procedure

Fresh spinach (Spinacia oleracea L.) leaves (ca. 1 kg)

↓ 0.001 M 1-litter EDTA (pH 8.0)
Homogenize in a warming blender
Filtrate with three layers of gauze cloth
Centrifuge at 4,000 rpm for 10 min.

↓ - Fraction I

Add 13.3 g of AmSO4/100 ml of sup. with gentle
↓ stirring
After 15 min., centrifuge at 4,000 rpm for 20 min., discard ppt
Add 15 g of AmSO4/100 ml of sup. with gentle
↓ stirring
After 15 min. centrifuge at 4,000 rpm for 20 min.
Dissolve ppt with 300 ml of 0.02 M phospate buffer (pH 6.0)

↓ - Fraction II

Adjust pH at 4.85 with addition of 1 N acetic acid 10-20 h under cold (4°C)
Centrifuge at 8,000 rpm for 20 min., discard ppt

↓ - Fraction III

Add 12.1 g of AmSO4/100 ml of sup. with gentle stirring
After 15 min., centrifuge at 8,000 rpm for 20 min., discard ppt
Add 7.9 g of AmSO4/100 ml of sup. with gentle stirring
After 15 min., centrifuge at 8,000 rpm for 20 min., discard sup.
Dissolve ppt with 0.001 M 100-ml EDTA (pH 7.9) - 0.0001 M 2-mercaptoethanol (for SH)

↓ - Fraction IV

Dialyze the sample against 5 l of the same buffer for 3 hr with 2 changes of buffer
↓ (discard ppt by centrifugation)
After dialysis, dilute the sample with equal volume of deionized water
Add 0.5 volume of calcium phosphate gel

↓ - Fraction V

Stir slowly for 15 min.
Centrifuge at 5,000 rpm for 15 min. and collect the gel
Elude enzyme from the gel with equal-volume sup. of 0.15 M phosphate buffer (pH 6.0)
↓ stir for 30 min.
Centrifuge at 5,000 rpm for 15 min., discard ppt

↓ - Fraction VI

Add 25 g of AmSO4/100 ml of sup.
After 30 min., centrifuge at 10,000 rpm for 20 min.
Dissolve ppt with small volume of 0.01 M phosphate buffer (pH 6.0)

↓ - Fraction VII

End

Table 1. Protein concentration in each fraction. C = concentration

    Fraction# Dilution  1st   2nd   3rd    C (mg/ml)
    I         ×   100  0.520 0.538 0.533   9.533
    II        ×   200  0.485 0.455 0.470  16.53
    III       ×   100  0.281 0.299 0.290   4.733
    IV        ×    40  0.304 0.320 0.312   2.052
    V         ×    10  0.380 0.390 0.385   0.660
    VI        ×    10  0.200 0.208 0.204   0.307
    VII       ×   100  0.210 0.208 0.209   3.133

II. Enzyme assay

Ly-hydroxypruvate 0.008 M (pH 6.0)  0.25 ml
NADH              1 mg/ml           0.2 ml
Ammonium sulfate  0.4 M             0.5 ml
Phosphate buffer  0.6 M (pH 6.0)    1.0 ml
Enzyme (sample)                       0.1 ml
Deionized water
Final volume                          3.1 ml

Enzyme dilution buffer: 0.01 M phosphate buffer (pH 6.0)
Results (Table 1)
I. Protein concentration in each fraction
II. Enzyme activity
When a sample is too dense to measure the concentration, re-measurement should be conducted after the dilution.
Measure NADH-NAD⁺ activity. Ex. NAD oxydase: NADH + O2 - NAD+ in 1 ml reactive solution (Table 2)
Measurement of Km
Fraction #VII was diluted to 1/2, 1/4, 1/8, 1/10, and 1/16, using 0.008 M substrates. Thereafter, measure the activities by the enzyme assay described above. (Results, omitted)
Thermal inactivation (熱失活)
Methods: Treat Fraction VII at 40°C for 10, 20, 40 and 80 min, and at 50°C for 1, 2, 3, 4, and 5 min. Measure the activities by enzyme assay method described above
Effect of salt on enzyme activity (塩の効果)
Methods: Measure reaction pace by various salts, i.e., KCl and NaCl, (0.4 M) with different soaking periods (0, 30, 60, 90, 120, 180, and 210 min., if possible soak longer time) and cmpare with (NH4)2SO4
Table 2. Results. SA = specific activity (units/ml·protein)

  Frac-  Volume  Protein  Activity   SA     Purifi-  Yield
  tion#  (ml)    (mg/ml)  (unit/ml)         cation   (%)
  I      232      9.533    5.114     0.5365   1.0  100.0
  II      50     16.53    19.27      1.1658   2.17  81.2
  III     45.5    4.733   18.625     3.9351   7.33  71.4
  IV      17      2.052   14.6       7.1150  13.26  20.9
  V       34      0.660    3.515     5.3258   9.93  10.1
  VI       ?      0.303    1.515     4.9349   9.20   ?
  VII      3      3.133   17.7       5.6495  10.53   4.5

Nitrogen

1. Quantitative analysis of nitrogen

UV absorbance = adjust at 280 nm Absorbance

Ex. Aromatic amino acid: phenylalanine: 250 nm = 190 mol Ab., Tryptophane: 278 nm = 5500 mol Ab., Trypsin: 275 nm = 1340 mol Ab.

2. Turbid method: acidic - low solubility

Sample: protein 0.5-1.5 mg/ml + 1.25% TCA (Trichloroacetic acid) 4 ml

3. Lowry's method: Phenol reagent

A: 2% Na2CO3 with 0.1N NaOH
B: 1% CuSO4·5H2O
C: 1% Na-tartrate or 1% K-tartrate
D: = B + C (1:1)
E: = A + D (50:1)

D and E should be prepared just before the using (within 24 hr), because those are unstable solution.

F: phenol reagent 1N

Procedure

sample (5-100 g protein) 0.6 ml
add 3.0 ml E ← agitation ← placing them for 10 min ← add 0.3 ml F ← agitation ← place them for 30 min
measuring at Ab750 (or Ab500)
Inhibitors on the measurement of Lawry's method: EDTA, AuSO4, Thial reagents, K⁺ ion, Glycerol, Triton X-100, etc.

4. Bradford's method (Analytical Biochemistry, 1976, 72, 248-252)

Dye

Dry reagent (ドライ試薬)

(Walter 1983)

Dry reagent chemistries in clinical analysis

Dry reagent chemistry

progress of quantitative measurments based on analytical chemistry on iatrochemistry

19c: invented litmus paper 1970s': developed dry reagent chemistry for measuring blood elements quantitatively

__Reagent zone__
_Reflective zone__
__Support zone___

Dry reagent chemistry: small amount of samples can be handled only by one step, monitored by small apparatus ← cheap

↔ Large amount of samples by several steps on many medical examinations

Components in dry reagent carrier

types of chemical reaction monitored
effectivness of supplied carrier

Fig. 1. Basic components of dry reagent chemistry carriers.

Enzyme immunoassay (酵素免疫測定法)

(Abbreviations: RIA = radioimmunoassay, FIA = fluorescent immunoassay, ELISA = enzyme-linked immuoassay, EMIT = enzyme-multiplied immunoassay)
Table 1. Immunoassay comparison

              RIA               FLA           EIA
Sensitivity   High (ng-pg)      High (ng-pg)  High (ng-pg)
Specificity   High              High          High
Speed         Days              Days          Hours
Reagents      Short shelf       Reasonable    Long shelf
                life              shelf life    life
Equipment     Scintillation or  UV monitor    Spectro-
    required    gamma counter                   photometer
Personnel     Skilled with        Skilled     Minimal
                license                         training
Cost          $7/test           $5/test       $2/test

Table 2. EIA comparison

ELISA ↔ EMIT
Heterogeneous assay ↔ Homogeneous assay
Reagent separation required (centrifugation or filtration) ↔ Reagent separation not required
Reagent washings required ↔ Step washings not required
Slower than EMIT ↔ Faster than ELISA
Sensitivity greater than EMIT ↔ Sensitivity less than ELISA
Macromolecules measured (antigens, antibodies) ↔ Measures small molecules (haptens*)
For diagnosing infectious diseases, immunoglobulins ↔ For drug, hormone, metabolite determinations
Solid phase assay ↔ Liquid phase assaya

* hapten (ハプテン, 付着体): small molecules that elicit an immune response only when attached to a large carrier such as a protein Ex. urushiol

__sample____________buffer/reagents__
_____↓___________________↓_____________sample handling
__diluter________________pipetter_____
_______________↓
__________test specimen__
_______________↓_______________________test measurements
________spectrophotometer__
_______________↓
_______absorbacne readings__

___________timer-printer___________
_____↓___________________↓
standard curve___test sample concentration___ data handling

Fig. 5. EMIT instrumentation

Polystyrene tube coated with antigen ← Incubate at 37°C ← Wash 3X ← Serum added ← Incubate at 37°C ← Wash 3X ← Enzyme-labeled antiserum added ← Incubate at 37°C ← Wash 3X ← Substrate added ← Visible color change ← Spectrophotometer absorption readings

Fig. 6. ELISA steps for serodiagnosis of diseases

Spectrophotometry (分光光度法)

High-sensitivity spectrophotometry

Table 1. Absorption detection limits in solution

Method_________________Detection limit (M)
Conventional transmission___1 × 10^-5
Laser intracavity___________5 × 10^-6
Interferometry_____________1 × 10^-7
Photothermal deflection_____1 × 10^-7
Photoacoustic_____________1 × 10^-7
Thermal lens______________8 × 10^-8

Table 2. Spectrophotometric methods

Transmission__________________Calorimetric
Conventional spectrophotometry__Thermocouple
Wavelength modulation_________Thermal lens
Laser intracavity_______________Phtoacoustic

Table 3. Criteria for comparison of spectrophotometric methods

Instrumental factors_____Sample factors
Tuning range__________Solvent physical properties
Pathlength effects_______Solvent absorbance
Reflection losses_______Molecular scatter
Flow effects___________Particulate scatter

Fig. 1. Experimental arrangement for filling and taking spectra with long hollow fibers. Source spectral light coupling correction is made by first recording a spectrum with a long fiber and then removing all but a small length and recording again. The difference is the transmission spectrum of the filled hollow fiber.
M1_______________________________M2
|– Gain – Reference loss – Samples loss –|– Detector
Fig. 2. Schematic configuration for quantitative intracavity absorption enhancement measurements. M1, M2 are mirrors.

Photon plumbing

Fiber optics (FO), waveguides, and evanescent waves as tools for chemical analysis
Optical wave guide (光導波路) - optic(al) fiber (fiberoptics, 光ファイバー)
Optical fiber: dielectric line for visible and near-infrared wavelengths

circular section of which refraction index is higher on the center than on the margin ← the light is walled in the fiber
flexible, transparent fiber made by drawing glass (silica) or plastic

Single mode fiber (単一モードファイバー): diameter of center (core) = several μm, used for long distance
Multimode fiber (多モードファイバー): diameter of center (core) = several tens μm, used for short distance

Fig. 1. The geometrical path of light passing through a section of optical fiber. The index of refraction of the surroundings, the fiber cladding, and the fiber itself are denoted by n₁, n₂, and n₃, respectively. If the path of the light shown enters at the greatest angle of acceptance of the fiber, then n₁sinθ = NA is the numerical aperture of the fiber. In terms of the modes, the case shown corresponds to a high-order mode (light entering far off-axis).

Fig. 2. The relationship between field intensity and transverse distance from the center of a symmetric waveguide is illustrated for the first three modes. The fields have an exponentially decreasing value beyond the surface of the waveguide. Outside the waveguide, the higher order modes have a greater intensity at a given distance than do lower order.
Fiber optics

Fig. 3. The path of a beam totally reflected within a prism and the associated evanescent field is illustrated. The index of refraction of the sample is less than that of the prism. In the lower part of the figure, the dependence of intensity on distance, y, from the interface is shown and the penetration depth, d_p, is indicated.

[ seed germination test ]

Culture (カルチャー)

cultivation, tillage (biology, including ecology)
the other meanings are omitted

Plant culture

Medium for water culture (hydroponics). Observation points: pH, leaf colors, and dry weight

	I	II	III	IV	V	IV
NaNO₃	0.75	0.75	0.75	-	0.75	-
CaCl₂·2H₂O	0.15	-	0.15	0.15	0.15	-
KH₂PO₄	0.25	0.25	-	0.25	0.25	-
MgSO₄·7H₂O	0.25	0.25	0.25	0.25	0.25	-
KCl	0.12	0.13	-	0.12	0.12	-
Fe₂(SO₄)₃ 2%	3 dp	3 dp	3 dp	3 dp	-	-
Na₂HPO₄	-	-	0.25	-	-	-
H₂O	1000	1000	1000	1000	1000	1000

Unit (単位)

International System of Units, SI (国際単位系)

The seven SI base units
kelvin (temperature)
second (time)
meter (length)
kilogram (mass)
candela (luminous intensity)
mole (amount of substance)
ampere (electric current)

Metric system (メートル法)

1 mm = 1/1000 m
1 μm = 1/1000 mm
1 nm = 1/1000 μm
1 Å = 1/10 nm

Cardinal numbers (基数)

100:____________ one hundred
101:____________ one hundred (and) one
1,000:___________one thousand
100,000:_________one hundred thousand
30,000,000,000:___three followed by ten zero
1,000,000:_______ one million
1,000,000,000:____one billion
1,000,000,000,000: one trillion
10¹⁵:____________one quadrillion
10¹⁸:____________one quintillion
10²¹:____________one sextillion
10²⁴:____________one septillion
10²⁷:____________one octillion
10³⁰:____________one nonillion
10³³:____________one decillion
10¹⁰: one followed by ten zeros (ten to the tenth)

Ratio

Percent vs percentage

Percent is a number.

"20 percent (20%) of people have pets". You cannot say "20 percentage of people have pets".

Percentage means a part of the whole.

"a small percentage of people have pets".

Per mille

1‰ = 10^-3 = 0.1% (⇒ isotope)

Validation (バリデーション, 検証)

An act, process, or instance of validating; especially : the determination of the degree of validity of a measuring device (Merrian-Webster Online)
Balance

Fig. Basic concept of the validation process like a balance.

Importance of validation

Technique: scientific principle useful for providing compositional information

Spectrophotometry

Procedure: Distinct adaptation of a technique for a selected measurement purpose

Pararosaniline method for measurement of sulfur dioxide

Method: Written directions necessary to use a method

ASTM D2914 - Standard Test Method for the Sulfur Dioxide Content of the Atmosphere (West Gaeke Method)

Protocol: Set of definitive directions that must be followed, without exception, if the analytical results are to be accepted for a given purpose

EPA Reference Method for the Determination of Sulfur Dioxide in the Atmosphere (Pararosaniline Method)

Fig. Collaborative test process.

Fig. 3. General process for evaluation/validation of methodology.